Human glycosylation enzymes

ABSTRACT

The present invention relates to novel human glycosylation enzyme polypeptides and isolated nucleic acids containing the coding regions of the genes encoding such polypeptides. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human glycosylation enzyme polypeptides. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human glycosylation enzyme polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/984,205, filed on Oct. 29, 2001 now U.S. Pat. No. 6,783,971 (issuedAug. 31, 2004), which is a divisional of U.S. application Ser. No.09/516,143, filed on Mar. 1, 2000 (now U.S. Pat. No. 6,333,182, issuedDec. 25, 2001), which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/122,409, filed on Mar. 2, 1999. Each ofthe above referenced patents and patent applications is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel human glycosylation enzymes. Morespecifically, isolated nucleic acid molecules are provided encodingthree enzymes involved in post-translational glycosylation: CMP SialicAcid Synthetase; Sialic Acid Synthetase and Aldolase, collectively“glycosylation enzymes.” Amino acid sequences comprising theglycosylation enzymes are also provided. The present invention furtherrelates to methods for treating physiologic and pathologic diseaseconditions, antibodies, and detection methods.

BACKGROUND OF THE INVENTION

During the last decade, numerous processes and procedures have beendeveloped for genetically engineering cells in order to produce a widevariety of proteins and glycoproteins. These procedures involveutilizing recombinant DNA technology to prepare a vector which includesgenetic material that codes for a specific protein or glycoprotein. Uponintroduction of the vector into the host cell, the inserted geneticmaterial instructs the host cell's biochemical machinery to manufacturea specific protein or glycoprotein.

Glycoproteins are proteins having carbohydrate groups attached atvarious points along the protein's amino acid backbone. The carbohydrategroups are commonly attached to asparagine, serine or threonine. Thegenetic sequence introduced into the host cell usually includesinstructions with respect to the amino acid sequence of the protein andthe location and structure of the carbohydrate groups. Most of the celllines which are commonly used as host cells are capable of following thevector's instructions with respect to preparing a protein having aspecific amino acid sequence. However, many host cells are not capableof following instructions with respect to glycosylation of the protein.For example, lepidopteran insect cells are a common host cell used inproducing a wide variety of proteins in a baculovirus system. However,such lepidopteran cells do not contain all of the cellular glycosylationmachinery present in mammalian cells required to attach certaincarbohydrate groups to the proteins it manufactures.

From the above, it is apparent that there is a need to identify humanpolypeptides which can be used to alter the glycosylation machinery ofnon-human host cells in order to control the structure of carbohydratesattached during glycosylation. Such a process for controlling host cellglycosylation would be useful not only in expressing glycoproteins whichaccurately mimic naturally occurring proteins, but would also be usefulin preparing glycoproteins having selected altered carbohydratestructures for diagnostic and research uses.

SUMMARY OF THE INVENTION

The present invention includes isolated nucleic acid moleculescomprising polynucleotides encoding a glycosylation enzyme polypeptide.The present invention further includes glycosylation enzyme polypeptidesencoded by these polynucleotides. The present invention further providesfor isolated nucleic acid molecules encoding portions (fragments) and/orvariants of full length glycosylation enzyme polypeptides and thepolypeptides encoded thereby.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a glycosylation enzyme polypeptide having an amino acidsequence as shown in the sequence listing; (b) a nucleotide sequenceencoding a mature glycosylation enzyme polypeptide having the amino acidsequences as shown in the sequence listing and described in Table 1; (c)a nucleotide sequence encoding a biologically active fragment of aglycosylation enzyme polypeptide having an amino acid sequence shown inthe sequence listing and described in Table 1; (d) a nucleotide sequenceencoding an antigenic fragment of a glycosylation enzyme polypeptidehaving an amino acid sequence shown in the sequence listing anddescribed in Table 1; (e) a nucleotide sequence encoding a glycosylationenzyme polypeptide comprising the complete amino acid sequence encodedby a human cDNA clone contained in the ATCC Deposit and described inTable 1; (f) a nucleotide sequence encoding a mature glycosylationenzyme polypeptide having an amino acid sequence encoded by a human cDNAclones contained in the ATCC Deposit and described in Table 1; (g) anucleotide sequence encoding a biologically active fragment of aglycosylation enzyme polypeptide having an amino acid sequence encodedby a human cDNA clone contained in the ATCC Deposit and described inTable 1; (h) a nucleotide sequence encoding an antigenic fragment of aglycosylation enzyme polypeptide having an amino acid sequence encodedby a human cDNA clone contained in the ATCC Deposit and described inTable 1; and (i) a nucleotide sequence complementary to any of thenucleotide sequences in (a) through (h), above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise, or alternatively consist of, a polynucleotidehaving a nucleotide sequence at least 80% identical, and more preferablyat least 85%, 90%, 95%, 97%, 98% or 99% identical, to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i)above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), (h), or (i), above. Polypeptides encoded by these nucleicacids or polynucleotides are also encompassed by the invention. Inspecific embodiments, polynucleotide which hybridizes to apolynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i) abovedoes not hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising,or alternatively consisting of, a polynucleotide which encodes the aminoacid sequence of an epitope-bearing portion of a glycosylation enzymepolypeptide having an amino acid sequence in (a), (b), (c), (d), (e),(f), (g), or (h), above. Polypeptides encoded by these nucleic acids arealso encompassed by the invention.

The present invention also relates to recombinant vectors, which includethe nucleic acid molecules of the present invention, and to host cellscontaining the recombinant vectors, as well as to methods of making suchvectors and host cells and for using them for production ofglycosylation enzyme polypeptides or peptides by recombinant techniques.Polypeptides produced by such methods are also provided.

In another embodiment, the invention provides isolated polypeptidescomprising a polypeptide having an amino acid sequence described in (a),(b), (c), (d), (e), (f), (g), or (h), above. Polypeptide portions(fragments) or variants of such glycosylation enzyme polypeptides arealso provided.

In a specific embodiment, the invention relates to a peptide orpolypeptide which comprises or alternatively consists of, the amino acidsequence of an epitope-bearing portion of a glycosylation enzymepolypeptide having an amino acid sequence described above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a glycosylation enzyme polypeptide of the invention includeportions of such polypeptides. In another embodiment, the inventionprovides an isolated antibody that specifically binds a glycosylationenzyme polypeptide having an amino acid sequence described above.

For a number of applications the level of glycosylation enzyme geneexpression can be detected in a sample of tissue or bodily fluid. Thepresence of glycosylation enzyme gene expression or an increased ordecreased level of glycosylation enzyme gene expression can be measured.Thus, the present invention provides for methods useful for detection ofglycosylation enzymes and for the diagnosis of applicable disorders. Thediagnosis of disorders involves assaying the expression level of thegene encoding the glycosylation enzyme protein in tissue or bodily fluidfrom an individual and comparing the gene expression level with astandard glycosylation enzyme gene expression level, whereby an increaseor decrease in the gene expression level over the standard is indicativeof a pathologic disorder.

DETAILED DESCRIPTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, the sequence identified as SEQ ID NO:1 wasgenerated by overlapping sequences contained in multiple clones (contiganalysis) corresponding to CMP Sialic Acid Synthetase. A representativeplasmid containing the sequence for SEQ ID NO:1 was deposited with theAmerican Type Culture Collection (“ATCC”). The sequence identified asSEQ ID NO:3 was also generated by contig analysis and corresponds toSialic Acid Synthetase coding sequences. A representative plasmidcontaining the sequence for SEQ ID NO:3 was deposited with ATCC.Additionally, the sequence identified as SEQ ID NO:5 was generated bycontig analysis and corresponds to Aldolase coding sequences. Arepresentative plasmid containing the sequence for SEQ ID NO:5 wasdeposited with ATCC.

As shown in Table 1, each clone is identified by a cDNA Clone ID(Identifier) and was deposited with the ATCC on Feb. 24, 2000 andassigned ATCC Deposit Number PTA-1410. The ATCC is located at 10801University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC depositwas made pursuant to the terms of the Budapest Treaty on theinternational recognition of the deposit of microorganisms for purposesof patent procedure.

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:1 (CMP Sialic Acid Synthetase), SEQID NO:3 (Sialic Acid Synthetase), or SEQ ID NO:5 (Aldolase) or a humancDNA contained within the plasmid HWLLM34, HA5AA37, or HDPAK85 depositedwith the ATCC. For example, the polynucleotide can contain thenucleotide sequence of the full length cDNA sequence, including the 5′and 3′ untranslated sequences, the coding region, with or without anatural or artificial signal sequence, the protein coding region, aswell as fragments, epitopes, domains, and variants of the nucleic acidsequence. Moreover, as used herein, a “polypeptide” refers to a moleculehaving the translated amino acid sequence generated from thepolynucleotide as broadly defined.

The CMP Sialic Acid Synthetase “polynucleotides” of the presentinvention also include portions (fragments) or variants of the sequencescontained in SEQ ID NO:1, the complement thereof, or the cDNA within theplasmid deposited with the ATCC, and portions (fragments) or variants ofthe polynucleotides encoding polypeptides of the invention (for example,polynucleotides encoding a polypeptide comprising or alternativelyconsisting of SEQ ID NO:2; CMP Sialic Acid Synthetase.) Polynucleotidesof the invention also include those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in for SEQ ID NO:1, the complements thereof, or the cDNAwithin the plasmid deposited with the ATCC.

The Sialic Acid Synthetase “polynucleotides” of the present inventioninclude portions (fragments) or variants of the sequences contained inSEQ ID NO:3, the complement thereof, or the cDNA within the plasmiddeposited with the ATCC, and portions (fragments) or variants of thepolynucleotides encoding polypeptides of the invention (for example,polynucleotides encoding a polypeptide comprising or alternativelyconsisting of SEQ ID NO:4; Sialic Acid Synthetase.) Polynucleotides ofthe invention also include those polynucleotides capable of hybridizing,under stringent hybridization conditions, to sequences contained in SEQID NO:3, the complements thereof, or the cDNA within the plasmiddeposited with the ATCC.

The Aldolase “polynucleotides” of the present invention include portions(fragments) or variants of the sequences contained in SEQ ID NO:5, thecomplement thereof, or the cDNA within the plasmid deposited with theATCC, and portions (fragments) or variants of the polynucleotidesencoding polypeptides of the invention (for example, polynucleotidesencoding a polypeptide comprising or alternatively consisting of SEQ IDNO:6; Aldolase.) Polynucleotides of the invention also include thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:5, the complementsthereof, or the cDNA within the plasmid deposited with the ATCC.

“Stringent hybridization conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly(A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, CMP Sialic AcidSynthetase, Sialic Acid Synthetase, and/or Aldolase polynucleotides canbe composed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the CMP Sialic Acid Synthetase, Sialic AcidSynthetase, and/or Aldolase polynucleotides can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. A CMPSialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of genomic flanking genes (i.e., 5′ or 3′ tothe CMP Sialic Acid Synthetase, Sialic Acid Synthetase, or Aldolasegenes of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolypeptides of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. CMP Sialic Acid Synthetase, Sialic AcidSynthetase, and/or Aldolase polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a CMP Sialic Acid Synthetase, SialicAcid Synthetase, and/or Aldolase polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/orAldolase polypeptide may contain many types of modifications. CMP SialicAcid Synthetase, Sialic Acid Synthetase, and/or Aldolase polypeptidesmay be branched, for example, as a result of ubiquitination, and theymay be cyclic, with or without branching. Cyclic, branched, and branchedcyclic CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/orAldolase polypeptides may result from posttranslation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a CMP Sialic Acid Synthetase polynucleotidesequence while “SEQ ID NO:2” refers to a CMP Sialic Acid Synthetasepolypeptide sequence as specified in Table 1.

“SEQ ID NO:3” refers to a Sialic Acid Synthetase polynucleotide sequencewhile “SEQ ID NO:4” refers to a Sialic Acid Synthetase polypeptidesequence as specified in Table 1.

“SEQ ID NO:5” refers to a Aldolase polynucleotide sequence while “SEQ IDNO:6” refers to a Aldolase polypeptide sequence as specified in Table 1.

Thus, the invention further includes CMP Sialic Acid Synthetase, SialicAcid Synthetase, and Aldolase polypeptide variants which show functionalactivity (e.g., biological activity). Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

A CMP Sialic Acid Synthetase, Sialic Acid Synthetase, or Aldolasepolypeptide “having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical, to CMPSialic Acid Synthetase, Sialic Acid Synthetase, or Aldolase enzymes ofthe present invention; including mature forms, as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof the CMP Sialic Acid Synthetase, Sialic Acid Synthetase, or Aldolasepolypeptide, but rather substantially similar to the dose-dependence ina given activity as compared to the to CMP Sialic Acid Synthetase,Sialic Acid Synthetase, or Aldolase polypeptide of the present invention(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity, and most preferably, not more than aboutthree-fold less activity relative to the CMP Sialic Acid Synthetase,Sialic Acid Synthetase, or Aldolase polypeptides of the presentinvention.)

Polynucleotides and Polypeptides of the Invention

Features of Protein Encoded by Gene No: 1

The translation product of this gene shares significant sequencehomology with mouse cytidine 5′-monophosphate N-acetylneuraminic acidsynthetase (See Genbank Accession No. AJ006215), which is thought to beimportant in the glycosylation of polypeptides. Based on the sequencesimilarity this gene has been termed “cytidine 5′-monophosphate sialicacid synthetase” or “CMP-Sialic Acid Synthetase” herein. Furthermore,based on the sequence similarity the translation product of this gene isexpected to share biological activities with mouse cytidine5′-monophosphate N-acetylneuraminic acid synthetase. Such activities canbe assayed using or routinely modifying methods known in the art, suchas, for example, those described in Proc. Natl. Acad. Sci. U S A (1998)95:9140-5, which is incorporated herein by reference in its entirety.

It has been discovered that this gene is expressed primarily in colontissue, and to a lesser extent in a variety of normal and transformedcell types.

Therefore, polynucleotides and polypeptides of the invention are usefulas reagents for differential identification of the tissue(s) or celltype(s) present in a biological sample. Similarly, polypeptides andantibodies directed to these polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the digestive system, expression of this gene atsignificantly higher or lower levels may be detected in certain tissues(e.g., colon and cancerous and wounded tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue from anindividual not having the disorder.

The homology to mouse cytidine 5′-monophosphate N-acetylneuraminic acidsynthetase suggests that polynucleotides and polypeptides correspondingto this gene are useful for treating a disease or condition resultingfrom the under expression of such a polypeptide in an individual.Polynucleotides and polypeptides corresponding to this gene may beuseful for the detection and/or treatment of disorders involvingaberrant glycolysis. Disorders involving aberrant glycolysis, resultingfrom aberrant activity of CMP Sialic Acid Synthetase or any other enzymeinvolved in the glycolytic pathway, typically manifests itself ascramps, myoglobinuria, or as an intolerance to exercise, or as a fixed,progressive weakness, to name a few. Additionally, polypeptides of theinvention and antibodies directed against these polypeptides may beuseful, for example, as a tumor marker and/or immunotherapy targets forthe above listed tissues.

Features of Protein Encoded by Gene No:2

The translation product of this gene shares significant sequencehomology with C. jejuni cytidine sialic acid synthetase (See GenbankAccession No. AJ000855), which is thought to be important in theglycosylation of polypeptides. Based on the sequence similarity thisgene has been termed “Sialic Acid Synthetase” herein. Furthermore, basedon the sequence similarity the translation product of this gene isexpected to share biological activities with C. jejuni cytidine sialicacid synthetase. Such activities can be assayed using or routinelymodifying methods known in the art.

The homology to C. jejuni cytidine sialic acid synthetase suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor treating a disease or condition resulting from the under expressionof such a polypeptide in an individual. Polynucleotides and polypeptidescorresponding to this gene may be useful for the detection and/ortreatment of disorders involving aberrant glycolysis. Disordersinvolving aberrant glycolysis, resulting from aberrant activity ofSialic Acid Synthetase or any other enzyme involved in the glycolyticpathway, typically manifests itself as cramps, myoglobinuria, or as anintolerance to exercise, or as a fixed, progressive weakness, to name afew. Additionally, polypeptides of the invention and antibodies directedagainst these polypeptides may be useful, for example, as a tumor markerand/or immunotherapy targets for tissues expressing these polypeptides.

Features of Protein Encoded by Gene No:3

The translation product of this gene shares sequence homology with E.coli N-acylneuraminic acid aldolase (See Nucleic Acids Res. 13 (24),8843-8852 (1985), incorporated herein by reference), which is a keyenzyme used by homofermentative bacteria involved in glycolysis. The endproduct of this pathway is lactic acid. Members of theheterofermentative bacteria include Streptococcus and Lactococcus, forexample. Based on the significant sequence similarity the translationproduct of this gene has been termed “Aldolase” herein. Furthermore,Aldolase is expected to share biological activity with E. coliN-acylneuraminic acid aldolase which activity can be assayed using orroutinely modifying methods known in the art.

It has been discovered that this gene is expressed primarily in immunecells and tissues such as primary dendritic cells, monocytes, and bonemarrow, and to a lesser extent in tonsilar tissue, B-cell lymphomas,spleen tissue, spinal cord tissue, and placental tissue.

Therefore, polynucleotides and polypeptides of the invention are usefulas reagents for differential identification of the tissue(s) or celltype(s) present in a biological sample and for diagnosis of diseases andconditions, including, but not limited to: muscular disorders involvedwith aberrant glycolysis, and immune system disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful toprovide immunological probes for differential identification of thesetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the muscular and immune systems,expression of this gene at significantly higher or lower levels may bedetected in certain tissues (e.g., muscular, immune, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid or spinal fluid) taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue from an individual not having thedisorder.

Given the homology to the E. coli aldolase protein, polynucleotides andpolypeptides corresponding to this are useful for the detection and/ortreatment of disorders involving aberrant glycolysis. Disordersinvolving aberrant glycolysis, resulting from aberrant activity ofaldolase or any other enzyme involved in the glycolytic pathway,typically manifests itself as cramps, myoglobinuria, or as anintolerance to exercise, or as a fixed, progressive weakness, to name afew. Additionally, polypeptides of the invention and antibodies directedagainst these polypeptides may be useful, for example, as a tumor markerand/or immunotherapy targets for the above listed tissues.

Additionally, each of the polypeptides described above are useful inmethods for making humanized proteins, e.g., the method described byBetenbough et al. in U.S. Provisional Patent Application Ser. No.60/122,582, filed Mar. 2, 1999, and by Betenbough et al. in U.S.Provisional Patent Application Ser. No. 60/169,624, filed Dec. 8, 1999,for expression of exogenous polypeptides in insect cells. U.S.Provisional Patent Application Ser. No. 60/122,582, filed Mar. 2, 1999and U.S. Provisional Patent Application Ser. No. 60/169,624, filed Dec.8, 1999 are hereby incorporated herein by reference in entirety. Thepolypeptides of the invention are responsible for glycosylation of otherpolypeptides. Such glycosylation is known to result in increasedretention of tertiary structure, increased resistance to proteases,half-life in blood, intermolecular interaction, and increasedsolubility. Many recombinantly produced therapeutically useful proteinsare known in the art; e.g., Human Growth Hormone and Alpha Interferon(to name just two), to which such technology could by applied to enhancetherapeutic properties of these proteins.

TABLE 1 ATCC NT 5′ NT 3′ NT 5′ NT AA Last Deposit SEQ Total of of of SEQAA Gene cDNA No: Z and ID NT Clone Clone Start ID of No. Clone ID DateVector NO: X Seq. Seq. Seq. Codon NO: Y ORF 1 HWLLM34 PTA-1410 pA2 11305 1 1305 1 2 434 (CMP Sialic Feb. Acid Synthetase) 24, 2000 2 HA5AA37PTA-1410 pA2 3 1080 1 1080 1 4 359 (Sialic Acid Feb. Synthetase) 24,2000 3 HDPAK85 PTA-1410 pA2 5 1429 1 1429 1 6 230 (Aldolase) Feb. 24,2000

Table 1 summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually three to five overlapping sequences at each nucleotideposition), resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID HWLLM34, HA5AA37, and HDPAK85 were deposited with theATCC on Feb. 24, 2000 and given ATCC Deposit No: PTA-1410. “Vector”refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq.” refers to the total number of nucleotides in the contigidentified by “Gene No.” The deposited clone contains all of thesesequences, reflected by the nucleotide position indicated as “5′ NT ofClone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative methionine start codon (ifpresent) is identified as “5′ NT of Start Codon.”

The translated amino acid sequence, beginning with the first translatedcodon of the polynucleotide sequence, is identified as “AA SEQ ID NO:Y,”although other reading frames can also be easily translated using knownmolecular biology techniques. The polypeptides produced by thesealternative open reading frames are specifically contemplated by thepresent invention.

SEQ ID NO:X and the translated SEQ ID NO:Y (where Y may be any of thepolypeptide sequences disclosed in the sequence listing) aresufficiently accurate and otherwise suitable for a variety of uses wellknown in the art and described further below. For instance, SEQ ID NO:Xis useful for designing nucleic acid hybridization probes that willdetect nucleic acid sequences contained in SEQ ID NO:X or the cDNAcontained in the deposited plasmid. These probes will also hybridize tonucleic acid molecules in biological samples, thereby enabling a varietyof forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used to generateantibodies which bind specifically to the proteins encoded by the cDNAclones identified in Table 1.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5, and the predicted translated aminoacid sequence identified as SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6,respectively, but also a sample of plasmid DNA containing a human cDNAof CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and Aldolasedeposited with the ATCC, as set forth in Table 1. The nucleotidesequence of each deposited plasmid can readily be determined bysequencing the deposited plasmid in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularplasmid can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedhuman cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,or the deposited clones. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include, but are not limited to, preparing probesor primers from the disclosed sequence and identifying or amplifying thecorresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:1, 2, 3, 4, 5, 6, or the deposited plasmids,using information from the sequences disclosed herein or the plasmidsdeposited with the ATCC. For example, allelic variants and/or specieshomologs may be isolated and identified by making suitable probes orprimers from the sequences provided herein and screening a suitablenucleic acid source for allelic variants and/or the desired homologue.

The CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolypeptides of the invention can be prepared in any suitable manner.Such CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolypeptides include isolated naturally occurring polypeptides,recombinantly produced polypeptides, synthetically producedpolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides are well understood inthe art.

The CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolypeptides may be in the mature form, or may be a part of a largerprotein, such as a fusion protein (see below). It is often advantageousto include an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purification,such as multiple histidine residues, or an additional sequence forstability during recombinant production.

The CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasepolypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques known in theart, such as, for example, the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). In specific embodiments, CMP Sialic AcidSynthetase, Sialic Acid Synthetase, and/or Aldolase polypeptides of theinvention are purified from natural or recombinant sources usingantibodies of the invention raised against CMP Sialic Acid Synthetase,Sialic Acid Synthetase, or Aldolase proteins using methods which arewell known in the art.

The present invention provides polynucleotides comprising, oralternatively consisting of, the nucleic acid sequences of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5 and/or a cDNA contained in the ATCC deposit.The present invention also provides polypeptides comprising, oralternatively, consisting of, the polypeptide sequences of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, and/or a polypeptide encoded by a cDNAcontained in the ATCC deposit. Polynucleotides encoding a polypeptidecomprising, or alternatively consisting of the polypeptide sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and/or a polypeptide sequenceencoded by a cDNA contained in the ATCC deposit are also encompassed bythe invention.

The following procedures can be used to obtain full length genes or fulllength coding portions of glycosylation enzyme genes using theinformation from the sequences disclosed herein or the clones depositedwith the ATCC.

Procedure 1

Race Protocol for Recovery of Full-length Genes

Partial cDNA clones can be made full-length by utilizing the rapidamplification of cDNA ends (RACE) procedure described in Frohman et al.Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missingeither the 5′ or 3′ end can be reconstructed to include the absent basepairs extending to the translational start or stop codon, respectively.In some cases, cDNAs are missing the start of translation, therefore.The following briefly describes a modification of this original 5′ RACEprocedure. Poly A+ or total RNA is reverse transcribed with SuperscriptII (Gibco/BRL) and an antisense or complementary primer specific to thecDNA sequence. The primer is removed from the reaction with a MicroconConcentrator (Amicon). The first-strand cDNA is then tailed with dATPand terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchorsequence is produced which is needed for PCR amplification. The secondstrand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoI, SalI and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removedcDNA is purified from the—agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such aspBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are available for purchase. Similarreagents and methods to those above are supplied in kit form fromGibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. Asecond kit is available from Clontech which is a modification of arelated technique, SLIC (single-stranded ligation to single-strandedcDNA), developed by Dumas et al. (Dumas et al., Nucleic Acids Res.,19:5227-5232 (1991)). The major differences in procedure are that theRNA is alkaline hydrolyzed after reverse transcription and RNA ligase isused to join a restriction site-containing anchor primer to thefirst-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that is difficult to sequencepast.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double-stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA clone. These methods include but are notlimited to filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′ RACE. While the full lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′ RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNA transcriptand a primer set containing a primer specific to the ligated RNAoligonucleotide and a primer specific to a known sequence of the gene ofinterest, is used to PCR amplify the 5′ portion of the desired fulllength gene which may then be sequenced and used to generate the fulllength gene. This method starts with total RNA isolated from the desiredsource, polyA RNA may be used but is not a prerequisite for thisprocedure. The RNA preparation may then be treated with phosphatase ifnecessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of the glycosylation enzyme gene ofinterest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the glycosylation enzymegene.

Polynucleotide and Polypeptide Fragments

The present invention is further directed to nucleic acid moleculesencoding portions or fragments of the polynucleotide sequences describedherein, e.g., shown in the sequence listing or contained in thedeposited clones. Uses for the polynucleotide fragments of the presentinvention include, but are not limited to, probes, primers, molecularweight, markers and for expressing the polypeptide fragments of thepresent invention. Fragments include portions of the polynucleotidesequences, at least 10 contiguous nucleotides in length selected fromany two integers, one of which representing a 5′ nucleotide position anda second of which representing a 3′ nucleotide position, where thefirst, or 5′ most, nucleotide for each disclosed polynucleotide sequenceis position 1. That is, every combination of a 5′ and 3′ nucleotideposition that a fragment at least 10 contiguous nucleotides in lengthcould occupy is included in the invention as an individual specie. “Atleast” means a fragment may be 10 contiguous nucleotide bases in lengthor any integer between 10 and the length of an entire nucleotidesequence minus 1. Therefore, included in the invention are contiguousfragments specified by any 5′ and 3′ nucleotide base positions of apolynucleotide sequences wherein the contiguous fragment is any integerbetween 10 and the length of an entire nucleotide sequence minus 1. Thepolynucleotide fragments specified by 5′ and 3′ positions can beimmediately envisaged using the clone description and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specifications. Although it is particularly pointed outthat each of the above described species are included in the presentinvention.

Further, the invention includes polynucleotides comprising sub-genusesof fragments specified by size, in nucleotides, rather than bynucleotide positions. The invention includes any fragment size, incontiguous nucleotides, selected from integers between 10 and the lengthof an entire nucleotide sequence minus 1 (where 1 is the first, or 5′most, nucleotide for each disclosed polynucleotide sequence). Preferredsizes of contiguous nucleotide fragments include 20 nucleotides, 30nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 125nucleotides, 150 nucleotides, 175 nucleotides, 200 nucleotides, 250nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides. Otherpreferred sizes of contiguous polynucleotide fragments, which may beuseful as diagnostic probes and primers, include fragments 50-300nucleotides in length which include, as discussed above, fragment sizesrepresenting each integer between 50-300. Larger fragments are alsouseful according to the present invention corresponding to most, if notall, of the polynucleotide sequences of the sequence listing ordeposited clones. The preferred sizes are, of course, meant to exemplifynot limit the present invention as all size fragments, representing anyinteger between 10 and the length of an entire nucleotide sequence minus1 of the sequence listing or deposited clones, are included in theinvention. Additional preferred nucleic acid fragments of the presentinvention include nucleic acid molecules encoding epitope-bearingportions of the polypeptides.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, or 1251to the end of SEQ ID NO:1, or the complementary strand thereto, or thecDNA contained in a deposited clone. In this context “about” includesthe particularly recited ranges, and ranges larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.Preferably, these fragments encode a polypeptide which has biologicalactivity. More preferably, these polynucleotides can be used as probesor primers as discussed herein. Polynucleotides which hybridize to thesenucleic acid molecules under stringent hybridization conditions or lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

Additionally, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, or 1051 to the end of SEQ ID NO:3, or thecomplementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

Also, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, or 1401 to the end of SEQ ID NO:5, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:2 orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340,341-360, 361-380, 381-400, 401-420, or 421 to the end of the codingregion of SEQ ID NO:2. Moreover, polypeptide fragments can be at leastabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, or 430 aminoacids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In the present invention, a “polypeptide fragment” also refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:4or encoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340,or 341 to the end of the coding region of SEQ ID NO:4. Moreover,polypeptide fragments can be at least about 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, or 350 aminoacids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In the present invention, a “polypeptide fragment” also refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:6or encoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, or 221 to the end of the coding region of SEQ ID NO:6.Moreover, polypeptide fragments can be at least about 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, or 220 amino acids in length. In this context “about” includes theparticularly recited ranges or values, and ranges or values larger orsmaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme orat both extremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The polynucleotide fragments, specified in contiguous nucleotides, canbe immediately envisaged using the above description and are thereforenot individually listed solely for the purpose of not unnecessarilylengthening the specification.

The present invention also provides for the exclusion of any fragment,specified by 5′ and 3′ base positions or by size in nucleotide bases asdescribed above for any nucleotide sequence of the sequence listing ordeposited clones. Any number of fragments of nucleotide sequencesspecified by 5′ and 3′ base positions or by size in nucleotides, asdescribed above, may be excluded from the present invention.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence contained in SEQ ID NO:Y or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion.

Fragments include portions of the amino acid sequences of the sequencelisting and encoded by deposited cDNA clones, at least 7 contiguousamino acid in length, selected from any two integers, one of whichrepresenting a N-terminal position and another representing a C-terminalposition. The first, or N-terminal most, codon of each polypeptidedisclosed herein is position 1. Every combination of a N-terminal andC-terminal position that a fragment at least 7 contiguous amino acidresidues in length could occupy, on any given amino acid sequence isincluded in the invention as an individual specie. At least means afragment may be 7 contiguous amino acid residues in length or anyinteger between 7 and the number of residues in a full length amino acidsequence minus 1. Therefore, included in the invention are species ofcontiguous fragments specified by any N-terminal and C-terminalpositions of amino acid sequence set forth in the sequence listing orencoded by the deposited cDNA clones, wherein the contiguous fragment isany integer between 7 and the number of residues in a full lengthsequence minus 1. The polypeptide fragments specified by N-terminal andC-terminal positions can be immediately envisaged using the abovedescription and are therefore not individually listed solely for thepurpose of not unnecessarily lengthening the specification. Although itis particularly pointed out that each of the above described species areincluded in the present invention.

Particularly, N-terminal deletions of the CMP Sialic Acid Synthetasepolypeptide can be described by the general formula m¹-434, where m¹ isan integer from 2 to 428, where m¹ corresponds to the position of theamino acid residue identified in SEQ ID NO:2. More in particular, theinvention provides polynucleotides encoding polypeptides comprising, oralternatively consisting of, a sequence selected from: D-2 to K-434; S-3to K-434; V-4 to K-434; E-5 to K-434; K-6 to K-434; G-7 to K-434; A-8 toK-434; A-9 to K-434; T-10 to K-434; S-11 to K-434; V-12 to K-434; S-13to K-434; N-14 to K-434; P-15 to K-434; R-16 to K-434; G-17 to K-434;R-18 to K-434; P-19 to K-434; S-20 to K-434; R-21 to K-434; G-22 toK-434; R-23 to K-434; P-24 to K-434; P-25 to K-434; K-26 to K-434; L-27to K-434; Q-28 to K-434; R-29 to K-434; N-30 to K-434; S-31 to K-434;R-32 to K-434; G-33 to K-434; G-34 to K-434; Q-35 to K-434; G-36 toK-434; R-37 to K-434; G-38 to K-434; V-39 to K-434; E-40 to K-434; K-41to K-434; P-42 to K-434; P-43 to K-434; H-44 to K-434; L-45 to K-434;A-46 to K-434; A-47 to K-434; L-48 to K-434; I-49 to K-434; L-50 toK-434; A-51 to K-434; R-52 to K-434; G-53 to K-434; G-54 to K-434; S-55to K-434; K-56 to K-434; G-57 to K-434; I-58 to K-434; P-59 to K-434;L-60 to K-434; K-61 to K-434; N-62 to K-434; I-63 to K-434; K-64 toK-434; H-65 to K-434; L-66 to K-434; A-67 to K-434; G-68 to K-434; V-69to K-434; P-70 to K-434; L-71 to K-434; I-72 to K-434; G-73 to K-434;W-74 to K-434; V-75 to K-434; L-76 to K-434; R-77 to K-434; A-78 toK-434; A-79 to K-434; L-80 to K-434; D-81 to K-434; S-82 to K-434; G-83to K-434; A-84 to K-434; F-85 to K-434; Q-86 to K-434; S-87 to K-434;V-88 to K-434; W-89 to K-434; V-90 to K-434; S-91 to K-434; T-92 toK-434; D-93 to K-434; H-94 to K-434; D-95 to K-434; E-96 to K-434; I-97to K-434; E-98 to K-434; N-99 to K-434; V-100 to K-434; A-101 to K-434;K-102 to K-434; Q-103 to K-434; F-104 to K-434; G-105 to K-434; A-106 toK-434; Q-107 to K-434; V-108 to K-434; H-109 to K-434; R-10 to K-434;R-11 to K-434; S-112 to K-434; S-113 to K-434; E-114 to K-434; V-115 toK-434; S-116 to K-434; K-117 to K-434; D-118 to K-434; S-119 to K-434;S-120 to K-434; T-121 to K-434; S-122 to K-434; L-123 to K-434; D-124 toK-434; A-125 to K-434; I-126 to K-434; I-127 to K-434; E-128 to K-434;F-129 to K-434; L-130 to K-434; N-131 to K-434; Y-132 to K-434; X-133 toK-434; N-134 to K-434; E-135 to K-434; X-136 to K-434; D-137 to K-434;I-138 to K-434; V-139 to K-434; G-140 to K-434; N-141 to K-434; I-142 toK-434; Q-143 to K-434; A-144 to K-434; T-145 to K-434; S-146 to K-434;X-147 to K-434; C-148 to K-434; L-149 to K-434; H-150 to K-434; P-151 toK-434; T-152 to K-434; D-153 to K-434; L-154 to K-434; Q-155 to K-434;K-156 to K-434; V-157 to K-434; A-158 to K-434; E-159 to K-434; M-160 toK-434; I-161 to K-434; R-162 to K-434; E-163 to K-434; E-164 to K-434;G-165 to K-434; Y-166 to K-434; D-167 to K-434; S-168 to K-434; X-169 toK-434; F-170 to K-434; S-171 to K-434; V-172 to K-434; V-173 to K-434;R-174 to K-434; R-175 to K-434; H-176 to K-434; Q-177 to K-434; F-178 toK-434; R-179 to K-434; W-180 to K-434; S-181 to K-434; E-182 to K-434;I-183 to K-434; Q-184 to K-434; K-185 to K-434; G-186 to K-434; V-187 toK-434; R-188 to K-434; E-189 to K-434; V-190 to K-434; T-191 to K-434;E-192 to K-434; P-193 to K-434; L-194 to K-434; N-195 to K-434; L-196 toK-434; N-197 to K-434; P-198 to K-434; A-199 to K-434; K-200 to K-434;R-201 to K-434; P-202 to K-434; R-203 to K-434; R-204 to K-434; Q-205 toK-434; D-206 to K-434; W-207 to K-434; D-208 to K-434; G-209 to K-434;E-210 to K-434; L-211 to K-434; Y-212 to K-434; E-213 to K-434; N-214 toK-434; G-215 to K-434; S-216 to K-434; F-217 to K-434; Y-218 to K-434;F-219 to K-434; A-220 to K-434; K-221 to K-434; R-222 to K-434; H-223 toK-434; L-224 to K-434; I-225 to K-434; E-226 to K-434; M-227 to K-434;G-228 to K-434; Y-229 to K-434; L-230 to K-434; Q-231 to K-434; G-232 toK-434; G-233 to K-434; K-234 to K-434; W-235 to K-434; H-236 to K-434;T-237 to K-434; T-238 to K-434; K-239 to K-434; C-240 to K-434; E-241 toK-434; L-242 to K-434; E-243 to K-434; H-244 to K-434; S-245 to K-434;V-246 to K-434; D-247 to K-434; I-248 to K-434; D-249 to K-434; V-250 toK-434; D-251 to K-434; I-252 to K-434; D-253 to K-434; W-254 to K-434;P-255 to K-434; I-256 to K-434; A-257 to K-434; E-258 to K-434; Q-259 toK-434; R-260 to K-434; V-261 to K-434; L-262 to K-434; R-263 to K-434;Y-264 to K-434; G-265 to K-434; Y-266 to K-434; F-267 to K-434; G-268 toK-434; K-269 to K-434; E-270 to K-434; K-271 to K-434; L-272 to K-434;K-273 to K-434; E-274 to K-434; I-275 to K-434; K-276 to K-434; L-277 toK-434; L-278 to K-434; V-279 to K-434; C-280 to K-434; N-281 to K-434;I-282 to K-434; D-283 to K-434; G-284 to K-434; C-285 to K-434; L-286 toK-434; T-287 to K-434; N-288 to K-434; G-289 to K-434; H-290 to K-434;I-291 to K-434; Y-292 to K-434; V-293 to K-434; S-294 to K-434; G-295 toK-434; D-296 to K-434; Q-297 to K-434; K-298 to K-434; E-299 to K-434;I-300 to K-434; I-301 to K-434; S-302 to K-434; Y-303 to K-434; D-304 toK-434; V-305 to K-434; K-306 to K-434; D-307 to K-434; A-308 to K-434;I-309 to K-434; G-310 to K-434; I-311 to K-434; S-312 to K-434; L-313 toK-434; L-314 to K-434; K-315 to K-434; K-316 to K-434; S-317 to K-434;G-318 to K-434; I-319 to K-434; E-320 to K-434; V-321 to K-434; R-322 toK-434; L-323 to K-434; I-324 to K-434; S-325 to K-434; E-326 to K-434;R-327 to K-434; A-328 to K-434; C-329 to K-434; S-330 to K-434; K-331 toK-434; Q-332 to K-434; T-333 to K-434; L-334 to K-434; S-335 to K-434;S-336 to K-434; L-337 to K-434; K-338 to K-434; L-339 to K-434; D-340 toK-434; C-341 to K-434; K-342 to K-434; M-343 to K-434; E-344 to K-434;V-345 to K-434; S-346 to K-434; V-347 to K-434; S-348 to K-434; D-349 toK-434; K-350 to K-434; L-351 to K-434; A-352 to K-434; V-353 to K-434;V-354 to K-434; D-355 to K-434; E-356 to K-434; W-357 to K-434; R-358 toK-434; K-359 to K-434; E-360 to K-434; M-361 to K-434; G-362 to K-434;L-363 to K-434; C-364 to K-434; W-365 to K-434; K-366 to K-434; E-367 toK-434; V-368 to K-434; A-369 to K-434; Y-370 to K-434; L-371 to K-434;G-372 to K-434; N-373 to K-434; E-374 to K-434; V-375 to K-434; S-376 toK-434; D-377 to K-434; E-378 to K-434; E-379 to K-434; C-380 to K-434;L-381 to K-434; K-382 to K-434; R-383 to K-434; V-384 to K-434; G-385 toK-434; L-386 to K-434; S-387 to K-434; G-388 to K-434; A-389 to K-434;P-390 to K-434; A-391 to K-434; D-392 to K-434; A-393 to K-434; C-394 toK-434; S-395 to K-434; Y-396 to K-434; A-397 to K-434; Q-398 to K-434;K-399 to K-434; A-400 to K-434; V-401 to K-434; G-402 to K-434; Y-403 toK-434; I-404 to K-434; C-405 to K-434; K-406 to K-434; C-407 to K-434;N-408 to K-434; G-409 to K-434; G-410 to K-434; R-411 to K-434; G-412 toK-434; A-413 to K-434; I-414 to K-434; R-415 to K-434; E-416 to K-434;F-417 to K-434; A-418 to K-434; E-419 to K-434; H-420 to K-434; I-421 toK-434; C-422 to K-434; L-423 to K-434; L-424 to K-434; M-425 to K-434;E-426 to K-434; K-427 to K-434; V-428 to K-434; and N-429 to K-434 ofSEQ ID NO:2. Polypeptides encoded by the polynucleotides are alsoencompassed by the invention.

N-terminal deletions of the Sialic Acid Synthetase polypeptide can bedescribed by the general formula m²-359, where m² is an integer from 2to 353, where m² corresponds to the position of the amino acid residueidentified in SEQ ID NO:4. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, a sequence selected from: P-2 to S-359; L-3 to S-359; E-4to S-359; L-5 to S-359; E-6 to S-359; L-7 to S-359; C-8 to S-359; P-9 toS-359; G-10 to S-359; R-11 to S-359; W-12 to S-359; V-13 to S-359; G-14to S-359; G-15 to S-359; Q-16 to S-359; H-17 to S-359; P-18 to S-359;C-19 to S-359; F-20 to S-359; I-21 to S-359; I-22 to S-359; A-23 toS-359; E-24 to S-359; I-25 to S-359; G-26 to S-359; Q-27 to S-359; N-28to S-359; H-29 to S-359; Q-30 to S-359; G-31 to S-359; D-32 to S-359;L-33 to S-359; D-34 to S-359; V-35 to S-359; A-36 to S-359; K-37 toS-359; R-38 to S-359; M-39 to S-359; I-40 to S-359; R-41 to S-359; M-42to S-359; A-43 to S-359; K-44 to S-359; E-45 to S-359; C-46 to S-359;G-47 to S-359; A-48 to S-359; D-49 to S-359; C-50 to S-359; A-51 toS-359; K-52 to S-359; F-53 to S-359; Q-54 to S-359; K-55 to S-359; S-56to S-359; E-57 to S-359; L-58 to S-359; E-59 to S-359; F-60 to S-359;K-61 to S-359; F-62 to S-359; N-63 to S-359; R-64 to S-359; K-65 toS-359; A-66 to S-359; L-67 to S-359; E-68 to S-359; R-69 to S-359; P-70to S-359; Y-71 to S-359; T-72 to S-359; S-73 to S-359; K-74 to S-359;H-75 to S-359; S-76 to S-359; W-77 to S-359; G-78 to S-359; K-79 toS-359; T-80 to S-359; Y-81 to S-359; G-82 to S-359; E-83 to S-359; H-84to S-359; K-85 to S-359; R-86 to S-359; H-87 to S-359; L-88 to S-359;E-89 to S-359; F-90 to S-359; S-91 to S-359; H-92 to S-359; D-93 toS-359; Q-94 to S-359; Y-95 to S-359; R-96 to S-359; E-97 to S-359; L-98to S-359; Q-99 to S-359; R-100 to S-359; Y-101 to S-359; A-102 to S-359;E-103 to S-359; E-104 to S-359; V-105 to S-359; G-106 to S-359; I-107 toS-359; F-108 to S-359; F-109 to S-359; T-110 to S-359; A-111 to S-359;S-112 to S-359; G-113 to S-359; M-114 to S-359; D-115 to S-359; E-116 toS-359; M-117 to S-359; A-118 to S-359; V-119 to S-359; E-120 to S-359;F-121 to S-359; L-122 to S-359; H-123 to S-359; E-124 to S-359; L-125 toS-359; N-126 to S-359; V-127 to S-359; P-128 to S-359; F-129 to S-359;F-130 to S-359; K-131 to S-359; V-132 to S-359; G-133 to S-359; S-134 toS-359; G-135 to S-359; D-136 to S-359; T-137 to S-359; N-138 to S-359;N-139 to S-359; F-140 to S-359; P-141 to S-359; Y-142 to S-359; L-143 toS-359; E-144 to S-359; K-145 to S-359; T-146 to S-359; A-147 to S-359;K-148 to S-359; K-149 to S-359; G-150 to S-359; R-151 to S-359; P-152 toS-359; M-153 to S-359; V-154 to S-359; I-155 to S-359; S-156 to S-359;S-157 to S-359; G-158 to S-359; M-159 to S-359; Q-160 to S-359; S-161 toS-359; M-162 to S-359; D-163 to S-359; T-164 to S-359; M-165 to S-359;K-166 to S-359; Q-167 to S-359; V-168 to S-359; Y-169 to S-359; Q-170 toS-359; I-171 to S-359; V-172 to S-359; K-173 to S-359; P-174 to S-359;L-175 to S-359; N-176 to S-359; P-177 to S-359; N-178 to S-359; F-179 toS-359; C-180 to S-359; F-181 to S-359; L-182 to S-359; Q-183 to S-359;C-184 to S-359; T-185 to S-359; S-186 to S-359; A-187 to S-359; Y-188 toS-359; P-189 to S-359; L-190 to S-359; Q-191 to S-359; P-192 to S-359;E-193 to S-359; D-194 to S-359; V-195 to S-359; N-196 to S-359; L-197 toS-359; R-198 to S-359; V-199 to S-359; I-200 to S-359; S-201 to S-359;E-202 to S-359; Y-203 to S-359; Q-204 to S-359; K-205 to S-359; L-206 toS-359; F-207 to S-359; P-208 to S-359; D-209 to S-359; I-210 to S-359;P-211 to S-359; I-212 to S-359; G-213 to S-359; Y-214 to S-359; S-215 toS-359; G-216 to S-359; H-217 to S-359; E-218 to S-359; T-219 to S-359;G-220 to S-359; I-221 to S-359; A-222 to S-359; I-223 to S-359; S-224 toS-359; V-225 to S-359; A-226 to S-359; A-227 to S-359; V-228 to S-359;A-229 to S-359; L-230 to S-359; G-231 to S-359; A-232 to S-359; K-233 toS-359; V-234 to S-359; L-235 to S-359; E-236 to S-359; R-237 to S-359;H-238 to S-359; I-239 to S-359; T-240 to S-359; L-241 to S-359; D-242 toS-359; K-243 to S-359; T-244 to S-359; W-245 to S-359; K-246 to S-359;G-247 to S-359; S-248 to S-359; D-249 to S-359; H-250 to S-359; S-251 toS-359; A-252 to S-359; S-253 to S-359; L-254 to S-359; E-255 to S-359;P-256 to S-359; G-257 to S-359; E-258 to S-359; L-259 to S-359; A-260 toS-359; E-261 to S-359; L-262 to S-359; V-263 to S-359; R-264 to S-359;S-265 to S-359; V-266 to S-359; R-267 to S-359; L-268 to S-359; V-269 toS-359; E-270 to S-359; R-271 to S-359; A-272 to S-359; L-273 to S-359;G-274 to S-359; S-275 to S-359; P-276 to S-359; T-277 to S-359; K-278 toS-359; Q-279 to S-359; L-280 to S-359; L-281 to S-359; P-282 to S-359;C-283 to S-359; E-284 to S-359; M-285 to S-359; A-286 to S-359; C-287 toS-359; N-288 to S-359; E-289 to S-359; K-290 to S-359; L-291 to S-359;G-292 to S-359; K-293 to S-359; S-294 to S-359; V-295 to S-359; V-296 toS-359; A-297 to S-359; K-298 to S-359; V-299 to S-359; K-300 to S-359;I-301 to S-359; P-302 to S-359; E-303 to S-359; G-304 to S-359; T-305 toS-359; I-306 to S-359; L-307 to S-359; T-308 to S-359; M-309 to S-359;D-310 to S-359; M-311 to S-359; L-312 to S-359; T-313 to S-359; V-314 toS-359; K-315 to S-359; V-316 to S-359; G-317 to S-359; E-318 to S-359;P-319 to S-359; K-320 to S-359; A-321 to S-359; Y-322 to S-359; P-323 toS-359; P-324 to S-359; E-325 to S-359; D-326 to S-359; I-327 to S-359;F-328 to S-359; N-329 to S-359; L-330 to S-359; V-331 to S-359; G-332 toS-359; K-333 to S-359; K-334 to S-359; V-335 to S-359; L-336 to S-359;V-337 to S-359; T-338 to S-359; V-339 to S-359; E-340 to S-359; E-341 toS-359; D-342 to S-359; D-343 to S-359; T-344 to S-359; I-345 to S-359;M-346 to S-359; E-347 to S-359; E-348 to S-359; L-349 to S-359; V-350 toS-359; D-351 to S-359; N-352 to S-359; H-353 to S-359; and G-354 toS-359 of SEQ ID NO:4. Polypeptides encoded by these polynucleotides arealso encompassed by the invention.

N-terminal deletions of the Aldolase polypeptide can be described by thegeneral formula m³-230, where m³ is an integer from 2 to 224, where m³corresponds to the position of the amino acid residue identified in SEQID NO:6. More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, asequence selected from: A-2 to N-230; F-3 to N-230; P-4 to N-230; K-5 toN-230; K-6 to N-230; K-7 to N-230; L-8 to N-230; Q-9 to N-230; G-10 toN-230; L-11 to N-230; V-12 to N-230; A-13 to N-230; A-14 to N-230; T-15to N-230; I-16 to N-230; T-17 to N-230; P-18 to N-230; M-19 to N-230;T-20 to N-230; E-21 to N-230; N-22 to N-230; G-23 to N-230; E-24 toN-230; I-25 to N-230; N-26 to N-230; F-27 to N-230; S-28 to N-230; V-29to N-230; I-30 to N-230; G-31 to N-230; Q-32 to N-230; Y-33 to N-230;V-34 to N-230; D-35 to N-230; Y-36 to N-230; L-37 to N-230; V-38 toN-230; K-39 to N-230; E-40 to N-230; Q-41 to N-230; G-42 to N-230; V-43to N-230; K-44 to N-230; N-45 to N-230; I-46 to N-230; F-47 to N-230;V-48 to N-230; N-49 to N-230; G-50 to N-230; T-51 to N-230; T-52 toN-230; G-53 to N-230; E-54 to N-230; G-55 to N-230; L-56 to N-230; S-57to N-230; L-58 to N-230; S-59 to N-230; V-60 to N-230; S-61 to N-230;E-62 to N-230; R-63 to N-230; R-64 to N-230; Q-65 to N-230; V-66 toN-230; A-67 to N-230; E-68 to N-230; E-69 to N-230; W-70 to N-230; V-71to N-230; T-72 to N-230; K-73 to N-230; G-74 to N-230; K-75 to N-230;D-76 to N-230; K-77 to N-230; L-78 to N-230; D-79 to N-230; Q-80 toN-230; V-81 to N-230; I-82 to N-230; I-83 to N-230; H-84 to N-230; V-85to N-230; G-86 to N-230; A-87 to N-230; L-88 to N-230; S-89 to N-230;L-90 to N-230; K-91 to N-230; E-92 to N-230; S-93 to N-230; Q-94 toN-230; E-95 to N-230; L-96 to N-230; A-97 to N-230; Q-98 to N-230; H-99to N-230; A-100 to N-230; A-101 to N-230; E-102 to N-230; I-103 toN-230; G-104 to N-230; A-105 to N-230; D-106 to N-230; G-107 to N-230;I-108 to N-230; A-109 to N-230; V-10 to N-230; I-111 to N-230; A-112 toN-230; P-113 to N-230; F-114 to N-230; F-115 to N-230; L-116 to N-230;K-117 to N-230; P-118 to N-230; W-119 to N-230; T-120 to N-230; K-121 toN-230; D-122 to N-230; I-123 to N-230; L-124 to N-230; I-125 to N-230;N-126 to N-230; F-127 to N-230; L-128 to N-230; K-129 to N-230; E-130 toN-230; V-131 to N-230; A-132 to N-230; A-133 to N-230; A-134 to N-230;A-135 to N-230; P-136 to N-230; A-137 to N-230; L-138 to N-230; P-139 toN-230; F-140 to N-230; Y-141 to N-230; Y-142 to N-230; Y-143 to N-230;H-144 to N-230; I-145 to N-230; P-146 to N-230; A-147 to N-230; L-148 toN-230; T-149 to N-230; G-150 to N-230; V-151 to N-230; K-152 to N-230;I-153 to N-230; R-154 to N-230; A-155 to N-230; E-156 to N-230; E-157 toN-230; L-158 to N-230; L-159 to N-230; D-160 to N-230; G-161 to N-230;I-162 to N-230; L-163 to N-230; D-164 to N-230; K-165 to N-230; I-166 toN-230; P-167 to N-230; T-168 to N-230; F-169 to N-230; Q-170 to N-230;G-171 to N-230; L-172 to N-230; K-173 to N-230; F-174 to N-230; S-175 toN-230; D-176 to N-230; T-177 to N-230; D-178 to N-230; L-179 to N-230;L-180 to N-230; D-181 to N-230; F-182 to N-230; G-183 to N-230; Q-184 toN-230; C-185 to N-230; V-186 to N-230; D-187 to N-230; Q-188 to N-230;N-189 to N-230; R-190 to N-230; Q-191 to N-230; Q-192 to N-230; Q-193 toN-230; F-194 to N-230; A-195 to N-230; F-196 to N-230; L-197 to N-230;F-198 to N-230; G-199 to N-230; V-200 to N-230; D-201 to N-230; E-202 toN-230; Q-203 to N-230; L-204 to N-230; L-205 to N-230; S-206 to N-230;A-207 to N-230; L-208 to N-230; V-209 to N-230; M-210 to N-230; G-211 toN-230; A-212 to N-230; T-213 to N-230; G-214 to N-230; A-215 to N-230;V-216 to N-230; G-217 to N-230; S-218 to N-230; F-219 to N-230; V-220 toN-230; S-221 to N-230; R-222 to N-230; D-223 to N-230; L-224 to N-230;and S-225 to N-230 of SEQ ID NO:6. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

Further, the invention includes polypeptides comprising sub-genuses offragments specified by size, in amino acid residues, rather than byN-terminal and C-terminal positions. The invention includes any fragmentsize, in contiguous amino acid residues, selected from integers between7 and the number of residues in a full length sequence minus 1.Preferred sizes of contiguous polypeptide fragments include at least 7amino acid residues, at least 10 amino acid residues, at least 20 aminoacid residues, at least 30 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 75 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, at least 150 amino acid residues, at least 175 amino acidresidues, at least 200 amino acid residues, at least 225 amino acidresidues, at least 250 amino acid residues, at least 275 amino acidresidues, at least 300 amino acid residues, at least 325 amino acidresidues, at least 350 amino acid residues, at least 375 amino acidresidues, at least 400 amino acid residues, at least 425 amino acidresidues, and at least 450 amino acid residues. The preferred sizes are,of course, meant to exemplify, not limit, the present invention as allsize fragments representing any integer between 7 and the number ofresidues in a full length sequence minus 1 are included in theinvention.

The contiguous polypeptide fragments specified by size in amino acidresidues of the present invention can be immediately envisaged using theabove description and are therefore not individually listed solely forthe purpose of not unnecessarily lengthening the specification.

The present invention also provides for the exclusion of any fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above. Any number of fragments specified byN-terminal and C-terminal positions or by size in amino acid residues asdescribed above.

It is particularly pointed out that the above fragments need not beactive since they would be useful, for example, in immunoassays, inepitope mapping, epitope tagging, to generate antibodies to a particularportion of the polypeptide, as vaccines, and as molecular weightmarkers.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotide fragments encoding these domains arealso contemplated.

Other preferred fragments are biologically active fragments. Biologicalactivity of fragments of the polypeptides of the invention can bedetermined using or routinely modifying assays known in the art.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of CMP Sialic Acid Synthetase,Sialic Acid Synthetase, and/or Aldolase polypeptides of the presentinvention. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 81:3998-4002.However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al. (1995) Mol. Gen. Genet.249:425-431. Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

A list of exemplified amino acid sequences comprising immunogenicepitopes of the invention are described elsewhere herein. It is pointedout that these descriptions only list amino acid residues comprisingepitopes predicted to have the highest degree of antigenicity using thealgorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186(said references incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis was performed using the computer programPROTEAN, using default parameters (Version 3.11 for the Power MacIntosh,DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Amino acidresidues comprising other immunogenic epitopes may be routinelydetermined using algorithms similar to the Jameson-Wolf analysis or byin vivo testing for an antigenic response using methods known in theart. See, e.g., Geysen et al., supra; U.S. Pat. Nos. 4,708,781;5,194,392; 4,433,092; and 5,480,971 (said references incorporated byreference in their entireties).

It is particularly pointed out that the described epitopic amino acidsequences comprise immunogenic epitopes. Thus, additional flankingresidues on either the N-terminal, C-terminal, or both N- and C-terminalends may be added to the sequences to generate an epitope-bearingpolypeptide of the present invention. Therefore, the immunogenicepitopes may include additional N-terminal or C-terminal amino acidresidues. The additional flanking amino acid residues may be contiguousflanking N-terminal and/or C-terminal sequences from the polypeptides ofthe present invention, heterologous polypeptide sequences, or mayinclude both contiguous flanking sequences from the polypeptides of thepresent invention and heterologous polypeptide sequences.

Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

The immuno and antigenic epitope-bearing fragments may be specified byeither the number of contiguous amino acid residues, as described above,or further specified by N-terminal and C-terminal positions of thesefragments on the amino acid sequence of SEQ ID NO:2, 4, or 6. Everycombination of a N-terminal and C-terminal position that a fragment of,for example, at least 7 or at least 15 contiguous amino acid residues inlength could occupy on the amino acid sequence of SEQ ID NO:2, 4, or 6is included in the invention. Again, “at least 7 contiguous amino acidresidues in length” means 7 amino acid residues in length or any integerbetween 7 amino acids and the number of amino acid residues of the fulllength polypeptide of the present invention. Specifically, each andevery integer between 7 and the number of amino acid residues of thefull length polypeptide are included in the present invention.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al., Proc. Natl. Acad. Sci. 82:5131-5135 at5134 (1985)).

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al., J. Gen. Virol. 66:2347-2354 (1985). If invivo immunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker suchas—maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carriers using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)Nature 331:84-86. Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem.270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotidesequences disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, thecomplementary strands thereto, and/or the cDNA sequences contained in adeposited clone.

The present invention also encompasses variants of the polypeptidesequences disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and/orencoded by a deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:1 or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:2, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions, or alternatively, under lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:2, the polypeptide sequenceencoded by the cDNA contained in a deposited clone, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein).

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:3 or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:4, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions, or alternatively, under lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:4, the polypeptide sequenceencoded by the cDNA contained in a deposited clone, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein).

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:5 or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:6, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions, or alternatively, under lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:6, the polypeptide sequenceencoded by the cDNA contained in a deposited clone, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence shown in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, the corresponding ORFs (open readingframes), or any fragments as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. (1990) 6:237-245). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences shown in the sequence listing or to the amino acidsequence encoded by a deposited cDNA plasmid, or a fragment thereof(e.g., as described herein) can be determined conventionally using knowncomputer programs. A preferred method for determining the best overallmatch between a query sequence (a sequence of the present invention) anda subject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

CMP Sialic Acid Synthetase, Sialic Acid Synthetase, and/or Aldolasevariants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993), reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1alphamutants that averaged 2.5 amino acid changes per variant over the entirelength of the molecule. Multiple mutations were examined at everypossible amino acid position. The investigators found that “[m]ost ofthe molecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes CMP Sialic Acid Synthetase, SialicAcid Synthetase, and Aldolase polypeptide variants which show functionalactivity (e.g., biological activity). Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

The present application is directed to CMP Sialic Acid Synthetasenucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequences disclosed herein, (e.g.,encoding a polypeptide having the amino acid sequence of an N and/or Cterminal deletion disclosed herein as m¹-n¹ of SEQ ID NO:2),irrespective of whether they encode a polypeptide having CMP Sialic AcidSynthetase functional activity. This is because even where a particularnucleic acid molecule does not encode a polypeptide having CMP SialicAcid Synthetase functional activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having CMP Sialic Acid Synthetase functional activityinclude, inter alia, (1) isolating a CMP Sialic Acid Synthetase gene orallelic or splice variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the CMP Sialic Acid Synthetase gene, asdescribed in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and (3) Northern Blotanalysis for detecting CMP Sialic Acid Synthetase mRNA expression inspecific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, which do, in fact, encode a polypeptidehaving CMP Sialic Acid Synthetase functional activity. By “a polypeptidehaving CMP Sialic Acid Synthetase functional activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to a functional activity of the CMP Sialic Acid Synthetase polypeptidesof the present invention (e.g., complete (full-length) CMP Sialic AcidSynthetase, mature (post-translationally modified) CMP Sialic AcidSynthetase and soluble CMP Sialic Acid Synthetase in a particularimmunoassay or biological assay as disclosed herein or otherwise knownin the art.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the nucleic acid sequence of adeposited cDNA, the nucleic acid sequence shown in SEQ ID NO:1, orfragments thereof, will encode polypeptides “having CMP Sialic AcidSynthetase functional activity.” In fact, since degenerate variants ofany of these nucleotide sequences all encode the same polypeptide, inmany instances, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving CMP Sialic Acid Synthetase functional activity. This is becausethe skilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

The present application is also directed to Sialic Acid Synthetasenucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequences disclosed herein, (e.g.,encoding a polypeptide having the amino acid sequence of an N and/or Cterminal deletion disclosed herein as m²-n² of SEQ ID NO:4),irrespective of whether they encode a polypeptide having Sialic AcidSynthetase functional activity. This is because even where a particularnucleic acid molecule does not encode a polypeptide having Sialic AcidSynthetase functional activity, one of skill in the art would still knowhow to use the nucleic acid molecule, for instance, as a hybridizationprobe or a polymerase chain reaction (PCR) primer. Uses of the nucleicacid molecules of the present invention that do not encode a polypeptidehaving Sialic Acid Synthetase functional activity include, inter alia,(1) isolating a Sialic Acid Synthetase gene or allelic or splicevariants thereof in a cDNA library; (2) in situ hybridization (e.g.,“FISH”) to metaphase chromosomal spreads to provide precise chromosomallocation of the Sialic Acid Synthetase gene, as described in Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and (3) Northern Blot analysis for detecting SialicAcid Synthetase mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, which do, in fact, encode a polypeptidehaving Sialic Acid Synthetase functional activity. By “a polypeptidehaving Sialic Acid Synthetase functional activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to a functional activity of the Sialic Acid Synthetase polypeptides ofthe present invention (e.g., complete (full-length) Sialic AcidSynthetase, mature (post-translationally modified) Sialic AcidSynthetase and soluble CMP Sialic Acid Synthetase in a particularimmunoassay or biological assay as disclosed herein or otherwise knownin the art.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the nucleic acid sequence of adeposited cDNA, the nucleic acid sequence shown in SEQ ID NO:3, orfragments thereof, will encode polypeptides “having Sialic AcidSynthetase functional activity.” In fact, since degenerate variants ofany of these nucleotide sequences all encode the same polypeptide, inmany instances, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving Sialic Acid Synthetase functional activity. This is because theskilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

The present application is also directed to Aldolase nucleic acidmolecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical tothe nucleic acid sequences disclosed herein, (e.g., encoding apolypeptide having the amino acid sequence of an N and/or C terminaldeletion disclosed herein as m³-n³ of SEQ ID NO:6), irrespective ofwhether they encode a polypeptide having Aldolase functional activity.This is because even where a particular nucleic acid molecule does notencode a polypeptide having Aldolase functional activity, one of skillin the art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having Aldolase functional activity include,inter alia, (1) isolating a Aldolase gene or allelic or splice variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe Aldolase gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); and (3)Northern Blot analysis for detecting Aldolase mRNA expression inspecific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, which do, in fact, encode a polypeptidehaving Aldolase functional activity. By “a polypeptide having Aldolasefunctional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to a functional activity of theAldolase polypeptides of the present invention (e.g., complete(full-length) Aldolase, mature (post-translationally modified) Aldolaseand soluble Aldolase in a particular immunoassay or biological assay asdisclosed herein or otherwise known in the art.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the nucleic acid sequence of adeposited cDNA, the nucleic acid sequence shown in SEQ ID NO:5, orfragments thereof, will encode polypeptides “having Aldolase functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having Aldolasefunctional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of the presentinvention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as anIgG Fc fusion region peptide, or leader or secretory sequence, or asequence facilitating purification. Such variant polypeptides are deemedto be within the scope of those skilled in the art from the teachingsherein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodieswhich are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. Monoclonal antibodies can be prepared using a wide oftechniques known in the art including the use of hybridoma andrecombinant technology. See, e.g., Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties). Fab and F(ab′)2 fragments may beproduced by proteolytic cleavage, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al.(1995) J. Immunol. Methods 184:177-186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene1879-18; Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864-869; and Sawai, H. et al. (1995) AJRI34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988) Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805-814; Roguska M. A. et al. (1994)PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645(said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994)Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies, S. O. et al.(1992) PNAS 89:1428-1432; Fell, H. P. et al. (1991) J. Immunol.146:2446-2452 (said references incorporated by reference in theirentireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng, B. et al. (1998) Blood 92(6):1981-1988; Chen, Z. et al.(1998) Cancer Res. 58(16):3668-3678; Harrop, J. A. et al. (1998) J.Immunol. 161(4):1786-1794; Zhu, Z. et al. (1998) Cancer Res.58(15):3209-3214; Yoon, D. Y. et al. (1998) J. Immunol.160(7):3170-3179; Prat, M. et al. (1998) J. Cell. Sci. 111(Pt2):237-247;Pitard, V. et al. (1997) J. Immunol. Methods 205(2):177-190; Liautard,J. et al. (1997) Cytokinde 9(4):233-241; Carlson, N. G. et al. (1997) J.Biol. Chem. 272(17):11295-11301; Taryman, R. E. et al. (1995) Neuron14(4):755-762; Muller, Y. A. et al. (1998) Structure 6(9):1153-1167;Bartunek, P. et al. (1996) Cytokine 8(1):14-20 (said referencesincorporated by reference in their entireties).

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because secreted proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgG), as described above, resulting inchimeric polypeptides. These fusion proteins facilitate purification andshow an increased half-life in vivo. One reported example describeschimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention can also be recovered from:products purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked. In addition, a methionine codon may be appropriatelyadded to vectors of the present invention, for the proper translation ofpolypeptides of the present invention which lack an N-terminalmethionine.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Both methods rely on binding of the polynucleotide to DNA or RNA. Forthese techniques, preferred polynucleotides are usually 20 to 40 basesin length and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense-Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an individual's genome. These sequences can beused to prepare PCR primers for amplifying and isolating such selectedDNA, which can then be sequenced. Using this technique, individuals canbe identified because each individual will have a unique set of DNAsequences. Once an unique ID database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, etc., can be amplified using PCR. In oneprior art technique, gene sequences amplified from polymorphic loci,such as DQa class II HLA gene, are used in forensic biology to identifyindividuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Oncethese specific polymorphic loci are amplified, they are digested withone or more restriction enzymes, yielding an identifying set of bands ona Southern blot probed with DNA corresponding to the DQa class II HLAgene. Similarly, polynucleotides of the present invention can be used aspolymorphic markers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as detection anddiagnostic probes for the presence of a specific mRNA in a particularcell type, as a probe to “subtract-out” known sequences in the processof discovering novel polynucleotides, for selecting and making oligomersfor attachment to a “gene chip” or other support, to raise anti-DNAantibodies using DNA immunization techniques, and as an antigen toelicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S.W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a detection or diagnostic method of adisorder, which involves (a) assaying the expression of a polypeptide ofthe present invention in cells or body fluid of an individual; (b)comparing the level of gene expression with a standard gene expressionlevel, whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof a marker for a cell type, cell condition, or disorder.

Moreover, polypeptides of the present invention can be used to treatdisease. For example, patients can be administered a polypeptide of thepresent invention in an effort to replace absent or decreased levels ofthe polypeptide (e.g., insulin), to supplement absent or decreasedlevels of a different polypeptide (e.g., hemoglobin S for hemoglobin B),to inhibit the activity of a polypeptide (e.g., an oncogene), toactivate the activity of a polypeptide (e.g., by binding to a receptor),to reduce the activity of a membrane bound receptor by absorbing freeligand (e.g., soluble TNF receptors used in reducing inflammation), orto bring about a desired response (e.g., blood vessel growth).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat disease. For example, administration of anantibody directed to a polypeptide of the present invention can bind andreduce levels of the polypeptide. Similarly, administration of anantibody can activate the polypeptide, such as by binding to apolypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Immune Activity

A polypeptide or polynucleotide of the present invention may be usefulin treating deficiencies or disorders of the immune system, byactivating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunedeficiencies or disorders may be genetic, somatic, such as cancer orsome autoimmune disorders, acquired (e.g., by chemotherapy or toxins),or infectious. Moreover, a polynucleotide or polypeptide of the presentinvention can be used as a marker or detector of a particular immunesystem disease or disorder.

A polynucleotide or polypeptide of the present invention may be usefulin treating or detecting deficiencies or disorders of hematopoieticcells.

A polypeptide or polynucleotide of the present invention could be usedto increase differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat thosedisorders associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

Moreover, a polypeptide or polynucleotide of the present invention couldalso be used to modulate hemostatic (the stopping of bleeding) orthrombolytic activity (clot formation). For example, by increasinghemostatic or thrombolytic activity, a polynucleotide or polypeptide ofthe present invention could be used to treat blood coagulation disorders(e.g., afibrinogenemia, factor deficiencies), blood platelet disorders(e.g. thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, a polynucleotide or polypeptide of thepresent invention that can decrease hemostatic or thrombolytic activitycould be used to inhibit or dissolve clotting. These molecules could beimportant in the treatment of heart attacks (infarction), strokes, orscarring.

A polynucleotide or polypeptide of the present invention may also beuseful in treating or detecting autoimmune disorders. Many autoimmunedisorders result from inappropriate recognition of self as foreignmaterial by immune cells. This inappropriate recognition results in animmune response leading to the destruction of the host tissue.Therefore, the administration of a polypeptide or polynucleotide of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders. For example,soluble forms of the polynucleotides of the present invention may beuseful in inhibiting cytokine activity by absorption.

Examples of autoimmune disorders that can be treated or detected by thepresent invention include, but are not limited to: Addison's Disease,hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a polypeptide or polynucleotide of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A polynucleotide or polypeptide of the present invention may also beused to treat and/or prevent organ rejection or graft-versus-hostdisease (GVHD). Organ rejection occurs by host immune cell destructionof the transplanted tissue through an immune response. Similarly, animmune response is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. The administrationof a polypeptide or polynucleotide of the present invention thatinhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, a polypeptide or polynucleotide of the present invention mayalso be used to modulate inflammation. For example, the polypeptide orpolynucleotide may inhibit the proliferation and differentiation ofcells involved in an inflammatory response. These molecules can be usedto treat inflammatory conditions, both chronic and acute conditions,including inflammation associated with infection (e.g., septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

A polypeptide or polynucleotide can be used to treat or detecthyperproliferative disorders, including neoplasms. A polypeptide orpolynucleotide of the present invention may inhibit the proliferation ofthe disorder through direct or indirect interactions. Alternatively, apolypeptide or polynucleotide of the present invention may proliferateother cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby a polynucleotide or polypeptide of the present invention include, butare not limited to neoplasms located in the: abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by a polynucleotide or polypeptide of the present invention.Examples of such hyperproliferative disorders include, but are notlimited to: hypergammaglobulinemia, lymphoproliferative disorders,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any otherhyperproliferative disease, besides neoplasia, located in an organsystem listed above.

Infectious Disease

A polypeptide or polynucleotide of the present invention can be used totreat or detect infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation and differentiationof B and/or T cells, infectious diseases may be treated. The immuneresponse may be increased by either enhancing an existing immuneresponse, or by initiating a new immune response. Alternatively, thepolypeptide or polynucleotide of the present invention may also directlyinhibit the infectious agent, without necessarily eliciting an immuneresponse.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by a polynucleotide orpolypeptide of the present invention. Examples of viruses, include, butare not limited to the following DNA and RNA viral families: Arbovirus,Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae,Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, encephalitis, eyeinfections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunisticinfections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the commoncold, Polio, leukemia, Rubella, sexually transmitted diseases, skindiseases (e.g., Kaposi's, warts), and viremia. A polypeptide orpolynucleotide of the present invention can be used to treat or detectany of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by a polynucleotide or polypeptideof the present invention include, but not limited to, the followingGram-Negative and Gram-positive bacterial families and fungi:Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea,Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus,Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,Syphilis, and Staphylococcal. These bacterial or fungal families cancause the following diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmphysema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. A polypeptide or polynucleotide of the presentinvention can be used to treat or detect any of these symptoms ordiseases.

Moreover, parasitic agents causing disease or symptoms that can betreated or detected by a polynucleotide or polypeptide of the presentinvention include, but not limited to, the following families:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), Malaria, pregnancycomplications, and toxoplasmosis. A polypeptide or polynucleotide of thepresent invention can be used to treat or detect any of these symptomsor diseases.

Preferably, treatment using a polypeptide or polynucleotide of thepresent invention could either be by administering an effective amountof a polypeptide to the patient, or by removing cells from the patient,supplying the cells with a polynucleotide of the present invention, andreturning the engineered cells to the patient (ex vivo therapy).Moreover, the polypeptide or polynucleotide of the present invention canbe used as an antigen in a vaccine to raise an immune response againstinfectious disease.

Regeneration

A polynucleotide or polypeptide of the present invention can be used todifferentiate, proliferate, and attract cells, leading to theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vascular (including vascularendothelium), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide of the present invention mayincrease regeneration of tissues difficult to heal. For example,increased tendon/ligament regeneration would quicken recovery time afterdamage. A polynucleotide or polypeptide of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated include of tendinitis, carpal tunnelsyndrome, and other tendon or ligament defects. A further example oftissue regeneration of non-healing wounds includes pressure ulcers,ulcers associated with vascular insufficiency, surgical, and traumaticwounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide of the present invention to proliferateand differentiate nerve cells. Diseases that could be treated using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic disorders (e.g., spinal corddisorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated using the polynucleotide or polypeptide of the presentinvention.

Chemotaxis

A polynucleotide or polypeptide of the present invention may havechemotaxis activity. A chemotaxic molecule attracts or mobilizes cells(e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells) to a particular sitein the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

A polynucleotide or polypeptide of the present invention may increasechemotaxic activity of particular cells. These chemotactic molecules canthen be used to treat inflammation, infection, hyperproliferativedisorders, or any immune system disorder by increasing the number ofcells targeted to a particular location in the body. For example,chemotaxic molecules can be used to treat wounds and other trauma totissues by attracting immune cells to the injured location. Chemotacticmolecules of the present invention can also attract fibroblasts, whichcan be used to treat wounds.

It is also contemplated that a polynucleotide or polypeptide of thepresent invention may inhibit chemotactic activity. These moleculescould also be used to treat disorders. Thus, a polynucleotide orpolypeptide of the present invention could be used as an inhibitor ofchemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., ligands andreceptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2): Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptide from suitablymanipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to a polypeptide of the invention comprising the steps of:(a) incubating a candidate binding compound with a polypeptide of theinvention; and (b) determining if binding has occurred. Moreover, theinvention includes a method of identifying agonists/antagonistscomprising the steps of: (a) incubating a candidate compound with apolypeptide of the invention, (b) assaying a biological activity, and(b) determining if a biological activity of the polypeptide has beenaltered.

Other Activities

A polypeptide or polynucleotide of the present invention may alsoincrease or decrease the differentiation or proliferation of embryonicstem cells, besides, as discussed above, hematopoietic lineage.

A polypeptide or polynucleotide of the present invention may also beused to modulate mammalian characteristics, such as body height, weight,hair color, eye color, skin, percentage of adipose tissue, pigmentation,size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide orpolynucleotide of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

A polypeptide or polynucleotide of the present invention may be used tochange a mammal's mental state or physical state by influencingbiorhythms, circadic rhythms, depression (including depressivedisorders), tendency for violence, tolerance for pain, reproductivecapabilities (preferably by Activin or Inhibin-like activity), hormonalor endocrine levels, appetite, libido, memory, stress, or othercognitive qualities.

A polypeptide or polynucleotide of the present invention may also beused as a food additive or preservative, such as to increase or decreasestorage capabilities, fat content, lipid, protein, carbohydrate,vitamins, minerals, cofactors or other nutritional components.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolatednucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a sequence of at least about 50 contiguous nucleotidesin the nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table 1.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of SEQ IDNO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Clone Sequence and ending withthe nucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table 1.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

Further preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 500 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizesunder stringent hybridization conditions to a nucleic acid molecule,wherein said nucleic acid molecule which hybridizes does not hybridizeunder stringent hybridization conditions to a nucleic acid moleculehaving a nucleotide sequence consisting of only A residues or of only Tresidues.

Also preferred is a composition of matter comprising a DNA moleculewhich comprises a human cDNA clone identified by a cDNA Clone Identifierin Table 1, which DNA molecule is contained in the material depositedwith the American Type Culture Collection and given the ATCC DepositNumber shown in Table 1 for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a humancDNA clone identified by a cDNA Clone Identifier in Table 1, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table 1.

Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said human cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid human cDNA containing the sequence of SEQ ID NO:X or contained inthe ATCC deposited clones.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical tosequence of at least 500 contiguous nucleotides in the nucleotidesequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is a method for detecting in a biologicalsample a nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table 1; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1; which method comprises a step of comparing a nucleotide sequence ofat least one nucleic acid molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidnucleic acid molecule in said sample is at least 95% identical to saidselected sequence.

Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

A further preferred embodiment is a method for identifying the species,tissue or cell type of a biological sample which method comprises a stepof detecting nucleic acid molecules in said sample, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1; and a nucleotide sequence encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1.

The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table 1, which methodcomprises a step of detecting in a biological sample obtained from saidsubject nucleic acid molecules, if any, comprising a nucleotide sequencethat is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table 1; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1.

The method for diagnosing a pathological condition can comprise a stepof detecting nucleic acid molecules comprising a nucleotide sequence ina panel of at least two nucleotide sequences, wherein at least onesequence in said panel is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1. The nucleic acid moleculescan comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:Y(wherein Y is any integer as defined in Table 1).

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the complete amino acid sequence ofSEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 7contiguous amino acids in the complete amino acid sequence of a proteinencoded by a human cDNA clone identified by a cDNA Clone Identifier inTable I and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of protein encoded bya human cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the protein encodedby a human cDNA clone identified by a cDNA Clone Identifier in Table 1and contained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the amino acid sequence of theprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

Further preferred is an isolated antibody which binds specifically to apolypeptide comprising an amino acid sequence that is at least 90%identical to a sequence of at least 7 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 7 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1; which method comprises a step of comparing an amino acid sequence ofat least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 7 contiguous amino acids.

Also preferred is the above method wherein said step of comparing anamino acid sequence of at least one polypeptide molecule in said samplewith a sequence selected from said group comprises determining theextent of specific binding of polypeptides in said sample to an antibodywhich binds specifically to a polypeptide comprising an amino acidsequence that is at least 90% identical to a sequence of at least 7contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table 1; and a complete amino acid sequence of a proteinencoded by a human cDNA clone identified by a cDNA Clone Identifier inTable 1 and contained in the deposit with the ATCC Deposit Number shownfor said cDNA clone in Table 1.

Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

Also preferred is a method for identifying the species, tissue or celltype of a biological sample which method comprises a step of detectingpolypeptide molecules in said sample, if any, comprising an amino acidsequence that is at least 90% identical to a sequence of at least 7contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table 1; and a complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

Also preferred is the above method for identifying the species, tissueor cell type of a biological sample, which method comprises a step ofdetecting polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 7 contiguous amino acids in a sequence selected from the abovegroup.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table 1, which methodcomprises a step of detecting in a biological sample obtained from saidsubject polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 7 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of aprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 7 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of aprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of aprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecule into a vector.Also preferred is the recombinant vector produced by this method. Alsopreferred is a method of making a recombinant host cell comprisingintroducing the vector into a host cell, as well as the recombinant hostcell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is ahuman protein comprising an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y, and an amino acidsequence of a protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1. The isolatedpolypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as liniting.

EXAMPLES Example 1 Isolation of a Selected cDNA Clone from the DepositedSample

Each cDNA clone in a cited ATCC deposit is contained in a pA2 plasmidvector (see Example 7). The deposited material in the sample assignedthe ATCC Deposit Number cited in Table 1 contains all of the plasmids ofTable 1. The ATCC deposit sample cited in Table 1 comprises a mixture ofapproximately equal amounts (by weight) of each different plasmid DNAs.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table 1. First,a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded bythe 5′ NT and the 3′ NT of the clone defined in Table 1) are synthesizedand used to amplify the desired cDNA using the deposited cDNA plasmid asa template. The polymerase chain reaction is carried out under routineconditions, for instance, in 25 μl of reaction mixture with 0.5 ug ofthe above cDNA template. A convenient reaction mixture is 1.5-5 mMMgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cyclesof PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min;elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetusautomated thermal cycler. The amplified product is analyzed by agarosegel electrophoresis and the DNA band with expected molecular weight isexcised and purified. The PCR product is verified to be the selectedsequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of a gene which may not be present in the depositedclone. These methods include but are not limited to, filter probing,clone enrichment using specific probes, and protocols similar oridentical to 5′ and 3′ “RACE” protocols which are well known in the art.For instance, a method similar to 5′ RACE is available for generatingthe missing 5′ end of a desired full-length transcript. (Fromont-Racineet al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the presentinvention is determined using protocols for Northern blot analysis,described by, among others, Sambrook et al. For example, a cDNA probeproduced by the method described in Example 1 is labeled with P³² usingthe rediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usingCHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70° C. overnight, and the films developed according to standardprocedures.

Example 4 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:X. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95° C.; 1 minute, 56°C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5minute cycle at 70° C. Human, mouse, and hamster DNA is used as templatein addition to a somatic cell hybrid panel containing individualchromosomes or chromosome fragments (Bios, Inc). The reactions isanalyzed on either 8% polyacrylamide gels or 3.5% agarose gels.Chromosome mapping is determined by the presence of an approximately 100bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI andinitiation/stop codons, if necessary, to clone the amplified productinto the expression vector. For example, BamHI and XbaI correspond tothe restriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D. ⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4° C. The cell debris is removed by centrifugation, and the supernatantcontaining the polypeptide is loaded onto a nickel-nitrilo-tri-aceticacid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc.,supra). Proteins with a 6×His tag bind to the Ni-NTA resin with highaffinity and can be purified in a simple one-step procedure (for detailssee: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferasegene as a selection marker, 2) an E. coli origin of replication, 3) a T5phage promoter sequence, 4) two lac operator sequences, 5) aShine-Delgarno sequence, and 6) the lactose operon repressor gene(lacIq). The origin of replication (oriC) is derived from pUC19 (LTI,Gaithersburg, Md.). The promoter sequence and operator sequences aremade synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells harvested by continuouscentrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of theexpected yield of protein per unit weight of cell paste and the amountof purified protein required, an appropriate amount of cell paste, byweight, is suspended in a buffer solution containing 100 mM Tris, 50 mMEDTA, pH 7.4. The cells are dispersed to a homogeneous suspension usinga high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4° C. overnight to allow furtherGuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 μm membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerseptiveBiosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pA2 is used to insert apolynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, XbaI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the cDNA sequence contained in the deposited clone isamplified using the PCR protocol described in Example 1 using primerswith appropriate restriction sites and initiation/stop codons. If thenaturally occurring signal sequence is used to produce the secretedprotein, the pA2 vector does not need a second signal peptide.Alternatively, the vector can be modified (pA2 GP) to include abaculovirus leader sequence, using the standard methods described inSummers et al., “A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures,” Texas Agricultural Experimental StationBulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five μg of a plasmid containing the polynucleotide is co-transfectedwith 1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μgof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27° C. The transfection solution is then removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

A polynucleotide of the present invention is amplified according to theprotocol outlined in Example 1 using primers with appropriaterestrictions sites and initiation/stop codons, if necessary. The vectorcan be modified to include a heterologous signal sequence if necessaryfor secretion. (See, e.g., WO 96/34891.)

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988)) The polypeptides can alsobe fused to heterologous polypeptide sequences to facilitate secretionand intracellular trafficking (e.g., KDEL). Moreover, fusion to IgG-1,IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector, and initiation/stop codons, if necessary.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and a polynucleotideof the present invention, isolated by the PCR protocol described inExample 1, is ligated into this BamHI site. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc Region

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGC (SEQ ID NO: 1)ACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGC CGCGACTCTAGAGGAT

Example 10 Formulating a Polypeptide

The polypeptide composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the secreted polypeptide alone), the site of delivery,the method of administration, the scheduling of administration, andother factors known to practitioners. The “effective amount” forpurposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofpolypeptide administered parenterally per dose will be in the range ofabout 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, asnoted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 μg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the polypeptide is typically administered at a dose rateof about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Pharmaceutical compositions containing the polypeptide of the inventionare administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray. “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The polypeptide is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed.Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped polypeptides. Liposomescontaining the secreted polypeptide are prepared by methods known perse: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, the polypeptide isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the polypeptideuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any polypeptide to be used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Polypeptides ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Example 11 Method of Treating Decreased Levels of the Polypeptide

It will be appreciated that conditions caused by a decrease in thestandard or normal expression level of a polypeptide in an individualcan be treated by administering the polypeptide of the presentinvention, preferably in the secreted and/or soluble form. Thus, theinvention also provides a method of treatment of an individual in needof an increased level of the polypeptide comprising administering tosuch an individual a pharmaceutical composition comprising an amount ofthe polypeptide to increase the activity level of the polypeptide insuch an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided in Example 10.

Example 12 Method of Treating Increased Levels of the Polypeptide

Antisense technology is used to inhibit production of a polypeptide ofthe present invention. This technology is one example of a method ofdecreasing levels of a polypeptide, preferably a secreted form, due to avariety of etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels of apolypeptide is administered intravenously antisense polynucleotides at0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The formulation of the antisense polynucleotide is provided in Example10.

Example 13 Method of Treatment Using Gene Therapy

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37° C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

1. An isolated protein comprising an amino acid sequence selected fromthe group consisting of: (a) amino acid residues 1 to 359 of SEQ IDNO:4; and (b) amino acid residues 2 to 359 of SEQ ID NO:4.
 2. Theprotein of claim 1, wherein the amino acid sequence is (a).
 3. Theprotein of claim 1, wherein the amino acid sequence is (b).
 4. Theprotein of claim 1 wherein the amino acid sequence further comprises aheterologous polypeptide.
 5. The protein of claim 1 wherein said proteinis glycosylated.
 6. The protein of claim 1 wherein said protein is fusedto polyethylene glycol.
 7. An isolated protein produced by a methodcomprising: (a) expressing the protein of claim 1 in a recombinant hostcell comprising a nucleic acid molecule encoding said protein; and (b)recovering the protein from the cell culture.
 8. A compositioncomprising the protein of claim 1 and a carrier.
 9. An isolated proteincomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length Sialic Acid Synthetasepolypeptide encoded by the HA5AA37 cDNA clone contained in ATCC DepositNo. PTA-1410; and (b) the amino acid sequence of the full-length SialicAcid Synthetase polypeptide, excluding the N-terminal methionineresidue, encoded by the HA5AA37 cDNA clone contained in ATCC Deposit No.PTA-1410.
 10. The protein of claim 9, wherein the amino acid sequence is(a).
 11. The protein of claim 9, wherein the amino acid sequence is (b).12. The protein of claim 9 wherein the amino acid sequence furthercomprises a heterologous polypeptide.
 13. The protein of claim 9 whereinsaid protein is glycosylated.
 14. The protein of claim 9 wherein saidprotein is fused to polyethylene glycol.
 15. An isolated proteinproduced by a method comprising: (a) expressing the protein of claim 9in a recombinant host cell comprising a nucleic acid molecule encodingsaid protein; and (b) recovering the protein from the cell culture. 16.A composition comprising the protein of claim 9 and a carrier.
 17. Anisolated polypeptide consisting of a fragment of SEQ ID NO:4, whereinsaid fragment is at least 30 contiguous amino acid residues in length.18. The polypeptide of claim 17 wherein the fragment is at least 50contiguous amino acid residues in length.
 19. The polypeptide of claim17 fused to a heterologous polypeptide.
 20. The polypeptide of claim 17wherein said protein is glycosylated.
 21. The polypeptide of claim 17wherein said protein is fused to polyethylene glycol.
 22. An isolatedpolypeptide produced by a method comprising: (a) expressing thepolypeptide of claim 17 in a recombinant host cell comprising a nucleicacid molecule encoding said polypeptide; and (b) recovering thepolypeptide from the cell culture.
 23. A composition comprising thepolypeptide of claim 17 and a carrier.
 24. An isolated polypeptideconsisting of a fragment of the full-length Sialic Acid Synthetasepolypeptide encoded by the HA5AA37 cDNA clone contained in ATCC DepositNo. PTA-1410, wherein said fragment is at least 30 contiguous amino acidresidues in length.
 25. The polypeptide of claim 24 wherein the fragmentis at least 50 contiguous amino acid residues in length.
 26. Thepolypeptide of claim 24 fused to a heterologous polypeptide.
 27. Thepolypeptide of claim 24 wherein said polypeptide is glycosylated. 28.The polypeptide of claim 24 wherein said polypeptide is fused topolyethylene glycol.
 29. An isolated polypeptide produced by a methodcomprising: (a) expressing the polypeptide of claim 24 in a recombinanthost cell comprising a nucleic acid molecule encoding said polypeptide;and (b) recovering the polypeptide from the cell culture.
 30. Acomposition comprising the polypeptide of claim 24 and a carrier.
 31. Anisolated polypeptide consisting of a fragment of the polypeptide of SEQID NQ:4 with sialic acid synthetase activity.
 32. The polypeptide ofclaim 31 wherein the polypeptide is fused to a heterologous polypeptide.33. The polypeptide of claim 31 wherein said protein is glycosylated.34. The polypeptide of claim 31 wherein said polypeptide is fused topolyethylene glycol.
 35. An isolated polypeptide produced by a methodcomprising: (a) expressing the polypeptide of claim 31 in a recombinanthost cell comprising a nucleic acid molecule encoding said polypeptide;and (b) recovering the polypeptide from the cell culture.
 36. Acomposition comprising the polypeptide of claim 31 and a carrier.